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d ultrasound energy can obviously improve plasmid DNA of pEGFP transfect efficiency in neovascularized corneal of rabbit.
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Objective To investigate whether pulsed ultrasound-mediated microbubbles destruction could effectively enhance the efficiency of liposome delivery plasmid to hepatoma cells.Methods The cultured HepG2 cells were divided into six groups.The first group was as contrast group and the second group was given proper dose of liposome and plasmid.Pulsed-ultrasound exposed liposome was the third group.Add plasmid and microbubbles to the fourth group and exposed to pulsed-ultrasound.Add plasmid and liposome to the fifth group and exposed to pulsed-ultrasound,too.Liposome with plasmid and microbubbles were applied to the sixth group and exposed to ultrasound.After 24 hours,the EGFP expression in the hepatoma cells was detected by fluorescence microscopy and MTT.Results Green fluorescence intensity and transfection efficiency of the fifth group were higher than that of other groups,and cell viability had no significant difference.Although they were high in the sixth group,cells were partly died.Conclusions The liposome delivered EGFP expression efficiency in hepatoma cells was increased with the administration of pulsed ultrasound-mediated microbubbles destruction.In certain condition,microbubbles can enhance the efficiency of plasmid.
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Objective To investigate the time-effect relationship of angiogenesis induced by ultrasound.mediated microbubble destruction in the skeletal muscle of rats.Methods Forty-eight healthy SD rats were divided into 4 groups:ultrasound-mediated microbubble destruction group,ultrasound only group,microbubble only group and control group.In ultrasound-mediated mierobubble destruction group, microbubbles were inj ected by vein at a dose of 0.5 ml and the target skeletal muscle was radiated at 2.0W/cm2.In ultrasound only group,the target skeletal muscle was radiated at 2.0 W/cm2.In microbubble only group.microbubbles were injected by vein at a dose of 0.5 ml.The control rats were without ultrasound radiation and microbubbles.On the 3rd,7th,10th,14th,21st and 28th day after the ultrasound radiation,two rats in each group were sacrificed and the target skeletal muscle was harvested for HE staining to observe the microstructure of tissue,immunohistochemistry staining was used to count the microvessel density (MVD),enzyme 1inked inmmunosorbent assay(ELISA)was used to detect the expression of vascular endothelial grouth factor(VEGF).Results Angiogenesis was significant in ultrasound-mediated mierobubhle destruction group,but a little in ultrasound only group.There was not any angiogenesis in either microbubble only group or control group.MVD and VEGF expression of ultrasound-mediated microbubble destruction group arrived at a peak on the 1Oth day as well as on the 14th day of ultrasound group.Conclusions Ultrasound-mediated microbubble destruction can facilliate the endogenous secretion of VEGF quickly and more,and accelerate the angiogenesis in the skeletal muscle.
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Objective Self-made docetaxel-carrying microbubbles were developed and evaluated as a new ultrasound contrast agent for diagnosis and targeted-chemotherapeutic drug delivery.Methods Docetaxel was mixed with an aqueous suspension of phospholipids in vials,which were put into 40℃ water for 30 minutes,after cooling,gas in vials was replaced with perfluoropropane gas,then vials were agitated for 45 s on a shaking device.Properties were studied contained concentration,size,zeta potential,drug entrapment efficiency and drug-loading amount.Drug released with ultrasound and imaging for VX2 carcinoma in rabbits were observed.Results Docetaxel-carrying microbubbles had a concentration of approximately 2.2×109～3.2×109/ml,a mean size of 623.1 am and a zeta potential of -(3.1±0.9)mV.Size distribution was 473.4～706.6 nm.Drug entrapment efficiency was more than 70% and drug-loading amount was(17.5±0.8)%.Fixed amount of ultrasound energy ruptured microbubbles and released docetaxel.Liver imaging of rabbits could be enhanced obviously and persistently,"fast in and out"phenomena was typical of VX2 carcinoma.Conclusions Liposome microbubbles represent a new class of acoustically active drug delivery vehicles.Docetaxel-carrying microbubbles can promote the value of ultrasound in tumor diagnosis,and docetaxel-carrying microbubbles combined with ultrasound have a hope to establish a kind of real-time monitoring,targeted system for tumor therapy.
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Objective To prepare the lipid ultrasound microbubbles carrying gene and transactivating transc"ptional activator(Tat)peptide and to study the efficiency of carrying gene and Tat as well as to study their ability as an ultrasound contrast agent.Methods The lipid ultrasound microbubbles were prepared using mechanical vibration method.The appearance,distribution,concentration,diameter and zeta potential were measured.The efficiencies of carrying gene and Tat were studyed by a confocal laser scanning microscopy and a fluorospectrophotometer.For the in vivo experiment,contrast-enhanced ultrasonography was performed on six rabbits to observe the duration and intensity of enhancement in the heart chambers and myocardium after the injection of the microbubbles.Results The diameter of the self-made lipid microbubbles carrying gene and Tat was(2.27±0.38)μm,the concentration was(3.07±0.42)×109/ml and zeta potential was(1.95±0.13)mV.The gene encapsulation efficiency for the lipid ultrasound microbubbles was 32%,and the Tat encapsulation efficiency was 35%.The in vivo experiment showed that the lipid ultrasound microbubbles could significantly enhance the echo intensity of myocardium.Conclusions The efficiency of carrying gene and Tat for the prepared lipid microbubbles is high,which can be used as a new vehicle carrying genes or drugs for therapy as well as an ultrasound contrast agent.
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Objective To develop perfluorocarbon lipid particles and investigate their basic properties,and target them to human hepatocellular carcinoma cells in vitro by hepatoma monocolonal antibody HAb18 with avidin-biotin interaction.Methods Rotary evaporation and high pressure homogen were used to prepare perfluorocarbon lipid particles, and the appearance and distribution of them were investigated by microscope and electron microscope, the concentration and the size and electric potential were detected.The biotinylated monoclonal antibody HAbl8 was prepared, then the biotinylated degree of the antibody was determined.The biotinylated perfluoroearbon lipid particles labelled with NBD were prepared and targeted to human hepatocellular carcinoma cells in vitro with avidin-biotin interaction.Results These perfluorocarbon lipid nanoparticles were uniform and stable,and the mean diameter of them was 171.9 nm.Hepatocellular carcinoma cells were surrounded by the biotinylated particles labelled with NBD.Conclusions A steady perfluoroearbon lipid particles were prepared and the biotinylated particles can be targeted to hepatocellular carcinoma cells with avidin-biotin interaction.
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Objective To obtain skin seed cells which can highly express active vascular endothelial growth factor 165(VEGF165) so as to promote refractory wound healing by gene therapy in combination with cell engineering.Methods After pIRES2-EGFP-hVEGF165 was transfected into dermal mesenchymal stem cells(DMSCs) by lipofectin,the expression of VEGF mRNA was detected by RT-PCR,VEGF protein in the supernatant and in the cells were assayed by enzyme-linked immunosorbent assay(ELISA) and Westen blotting respectively.To evaluate the activity of VEGF secreted by transfected DMSCs,hECV304 was cultured with the supernatant of transfected DMSCs and its proliferation activity was analyzed by MTT assays.Meanwhile,the proliferation activity of VEGF-transfected DMSCs and non transfected DMSCs was investigated by MTT.Results The results of RT-PCR,ELISA and Western blot demonstrated that the expression of VEGF in transfected DMSCs was about 1.6 times than that of control DMSCs.The product not only enhanced the proliferation of hECV304 but also increased the proliferation of transfected DMSCs.Conclusion The plasmid pIRES2-EGFP-hVEGF165 is successfully transfected into DMSCs with the aid of lipotransfection,and hVEGF165-transfected DMSCs might high-efficiently secrete highly active VEGF165.
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Objective To explore photodynamic inhibition of pyropheophorbide-a methyl ester(MPPa) in nasopharyngeal carcinoma cells and the mechanism.Methods The poorly differentiated CNE2 cell line was chosen to investigate the photocytotoxic potency and efficacy of MPPa 24 h after PDT by MTT reduction assay.The apoptosis of CNE2 cells was analyzed 8 h after photosensitization of MPPa(2 ?mol/L) under red light irradiation at a light dose of 2 J/cm~(2) by flow cytometry with Annexin Ⅴ-FITC and PI staining and nuclear staining with Hoechst 33258,and the changes in mitochondrial membrane potential(△?m) were monitored by flow cytometry with Rhodamine123 staining. Results Drug-and light-dose response curves were observed,showing that MPPa mediates a dose-and light-dependent phototoxicity in the NPC/CNE2 cells.MPPa could significantly trigger the NPC/CNE2 cells into apoptosis and the collapse of △?m were observed in the NPC/CNE2 cells treated by PDT with MPPa.Conclusion MPPa can effectively photokill the NPC/CNE2 cells and induce apoptosis as a prominent mode of cell death probably through mitochondria-initiated apoptosis pathway.
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Objective To study on the mechanisms that photodynamic therapy with laser-activated BPD-MA induces human bladder cancer BIU-87 cell apoptosis.Methods Human bladder cancer BIU-87 cells at log phase were randomized into 4 groups.The cells of BDP-MA-PDT groups were added with BPD-MA and then activated with 632.8 nm He-Ne laser;PDT group without BDP-MA addition was only irradiated;BDP-MA group without irradiation;normal control group without BDP-MA addition or irradiation.Photosensitization of BPD-MA was activated by laser with red light(632.8 nm) delivered at 10 mw/cm~(2) to give a total dose of 2.4 J/cm~(2).The expression of Bcl-2 and Bax proteins in human bladder cancer BIU-87 cells were detected by immunohistochemical staining.Results The Bcl-2 protein expression in BPD-MA-PDT group was lower than that of other groups(P0.05),therefore the ratio of Bax to Bcl-2 obviously increased(P